MULTIPLICATION AND EVALUATION OF TISSUE CULTUREDERIVED WALNUT PLANTS

Gale McGranahan, Walt Tulecke, Chuck Leslie

ABSTRACT

The purpose of this project was to use and improve current walnuttissue culture methodology to generate plants from somatic embryosand from mature Juglans regia cultivars. Ninty-eight somatic embryoderived plants are now in soil. Fiftyfour of these are derived fromthe same embryogenic line. Plants from the same line are phenotypi-cally uniform with the exception of 15% which exhibit thickened leaves,and abnormal bud development. This variant does not appear to beassociated with lack of chilling. Modifications in the procedurefor obtaining plants from embryogenic lines has improved the rateof survival from embryo to potted plant from 5 to 13%. Modificationsincluded omission of cold treatment, pretreatment of selected embryoswith GA3 and addition of activated charcoal to basal medium usedfor germlnation.

Ten mature cuItivars were initiated into culture. Chandler is nowin the multiplication phase and Sunland and Vina are approachingthat phase. Problems encountered in micropropagation of mature J.regia cultivars included inadequate explant sterilization, latentcontamination and 'slow decline'. Slow decline is characterizedby gradual leaf chlorosis, leaf abscission and eventual death. Inade-quate sterilization was overcome by rinsing explants for 2 hrs inrunning tap water prior to sterilization and by using individualrather than batch sterilization procedures. Antibiotic supplementsto the medium were not effective in elminating contamination. Slowdecline, which may be a result of lack of juvenility, was unaffectedby several media adjustments. It was somewhat overcome by rapidtransfer.

OBJECTIVE

This project had 2 main objectives. The first was to use the technol-ogy developed for somatic embryogenesis to obtain sufficient numbersof somatic embryo derived walnut plants to determine whether somaclonalvariation will be an important factor in genetic improvement of wal-nuts. The second was to develop the methodology to micropropagatemature Persian walnut cultivars on their own roots.

PROCEDURE

A. Somatic Embryo Derived Plants

A procedure for obtaining somatic embryos was reported briefly inWalnut Research Reports, 1984 and in detail in Plant Science (Tulecke,W. and G. McGranahan, 1985. Somatic embryogenesis and plant regenera-tion from cotyledons of walnut, Juglans regia L. Plant Science 4:57-63). The procedure is summarized in Table 1.

Very few of the original selected embryos survive the entire procedure

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to become established plants. Major losses occur due to embryo deathin cold storage, failure to develop roots and shoots and failureto survive transplanting to soiL In order to efficiently obtainsomatic embryo derived plants modifications in the established proce-dure were examined. These included omission of the cold treatment,media adjustments to enhance root and apex developOment and changesin the soil transfer procedures. These are described in resultsand discussion0 The repetitively embryogenic line SFA was used inall studies.

B. Micropropagation of Mature Persian Cultivars

Procedures for micropropagating Juglans have been described (McGrana-han, G., J. Driver and Wo Tulecke. Tissue Culture of Juglans in:Bonga J. and D. Durzan (eds) Tissue Culture in Forestry Vol. 2 MartinusNi jhoff , Netherlands (in press)). The methods described have beensuccessfully applied to juvenile J. regia, J. hindsii Iparadox' andpterocarya sp, to mature 'paradox' and to suckers of mature Jo hindsii.Results with mature J. regia have been inconsistent and largely unsuc-cessful. Problems encountered in culturing mature J. regia includedifficulties in obtalning sterile explant material, 'latent' contamina-tion possibly from internal contaminants, and 'slow decline' dueto unknown causes. Slow decline is characterized by gradual chlorosis,leaf abscission and death. To overcome these problems, modificationsin the sterilization procedure (including use of antibiotics, Rifampi-cin (100 mg/ml) and Tetracycline (100 mg/ml), frequency of transfer,timing of excision of shoots from original explants and hormone levels(BAP at 2,5 mg/l) were investigated. Ten cultivars were used (Sun-land, Vina, Eureka, Chandler, Howard, Amigo, Scharsch Franquette,Chico, Hartley and 66-178). All material had been grafted on tojuvenile rootstock (2nd leaf) and half had been sprayed with BAP(100 mg/l) and GA3 (50 mg/l) at weekly intervals and repeatedly cutback as previously descrlbed (McGranahan et. al.).

RESULTS AND DISCUSSION

A. Somatic Embryo Derived Plants

A total of 504 embryos were selected from the SF4 repetitively embryo-genic culture line0 288 were given cold treatment (8-10 wks at 4°C,dark) and 216 were given no cold treatment. Omission of cold treat-ment. increased the number of embryos that germinated and survivedfrom 5% to 13% (Table 2)0 Eight of the total surviving plants were"deformed". They exhibited thickened leaves and abnormal buds.Initially, these symptoms were thought to be a result of lack ofchilling but 3/8 of the deformed plants had been given the standardcold treatment.

Rooting efficiency declined with removal of cold treatment. Severalchanges in media and embryo handling were examined to correct thisproblem. Of these, placement of selected embryos on basal medj,..y.:m...supplemented with 20 mg/l GA3 for 1 week in the dark immediatelyprior to transfer to light, was the most effective for root initiation.This procedure increased rooting frequency to 75%, approximatelya 4 fold increase 0 Root extension growth and quality was markedly

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improved by addition of 5% charcoal to the basal medium used forroot growth in the light.

Reliable apex development and shoot extension remains a serious obsta-cle to efficient production and has not been satisfactorily correctedby either media adjustments or culturing procedures.

Survival during transfer from steriIe in vitro conditions to non-sterile soil was also low (10%)0 Soil sterilization, fumgicide appli-cations, and selection for increased root size prior to transplantfailed to impact this problem. By selecting only plants with stemelongation and/or leaf development in vitro, transfer survival wasincreased to 66%. This reinforces the importance of apex developmentin vitro.

Currently, 98 somatic embryo derived plants are in soil. Their originsare listed in Table 3.

Our next objective is to determine if varlation is present in theseplants. This may be evaluated through isozyme analysis, chromosomecounts and replicated trials. Heritable variation can only be assessedby selfing plants and evaluating progeny. The plants we have nowappear phenotypically uniform with the exception of the deformedplants.

B. Micropropagation of Mature Cultivars.

Explants were collected 11 tlmes between 2/12/85 and 10/18/85. Eachcultivar was collected at least tWlce with average of 44 explantsper collection. Vina, Chandler and Sunland were collected 7, 5 and4 times respectively. Percent survival after 2 months in culturewas increased from 3% to 58% in Vina by altering sterilization methodfrom batch to individual sterilizatlon and rinsing for 2-3 hrs inrunning tap water prior to sterilizationo Antibiotics were effectivein increasing survival at 2 wks from 61% (SE = 4.9) to 91% (SE =1.8). However, the antibiotics produced increasing phytoxicity after3 days and when explants were removed from media with antibiotics,contamination eventually reappeared.

The slow decline which may be a symptom of a suboptimal medium wasnot positively influenced by raisln BAP levels (2x and 5x standard),addition of charcoal to mediurn,or reducing medlurnto half strength.Treatments that had a positlve effect on long term survival includedmaintaining the shoot on the original explant as long as possiblebefore excision and rapld transfer (transfer to fresh medium everyother day).

At present Chandler is in multiplication phase and Sunland and Vinaare approaching multiplication phase. Explants of the latter twoclones have been dlstributed to Plant Research Laboratory.

Many questions remain to be answered in micropropagation of matureJ. regia cultivars. Because we, have been successful in obtainingapparently clean explants we feel the major problem interfering withmicropropagation is slow decline. Slow decline, characterized by

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gradual leaf yellowing and abscission followed by microplant death,has been noted only in mature cultivars. If slow decline is relatedto lack of juvenility it could be attacked through several routes,the first being rejunvenation in vivo. Serial grafting or stimulationof growth through GA/BAP sprays and pruning are possibilities thathave been successful in other crops. To obtain more than 2 generationsof grafting/year, techniques for grafting in the greenhouse on veryyoung seedlings would have to be developed 0 A better method forrejunvenation in vivo would be the development of apomictic seedlings.This would alsO-provide material to study the basic differences betweenjuvenile and mature material of the same cultivar. (Dr. Vito Politoplans to submit a proposal on apomixis in walnuts.) Rejuvenationin vitro is another potential means of overcoming slow decline. Serialmicrografting onto multiplying J. regia (juvenile) or paradox shootsin culture or onto developing embryos (derived through somaticembryogensis or sterile nut culture) appears feasible. J. Driverof Plant Research Laboratory has indicated a willingness to pursuein vitor rejuvenation. Embryogenic cultures and juvenile J. regiacultures have been released to him for this purpose. The final optionfor micropropagating mature cultivars is to develop media specificfor mature cultivars. This would require developing matrices ofnumerous media components. There is potential of success using thismethod but the time and-labor involved are prohibitive at this time.

FUTURE RESEARCH DIRECTIONS

Somatic embryogenesls is a powerful tool in genetically improvingwalnuts. The potential of somaclonal variation will be unknown untilplants derived from somatic embryos are thoroughly evaluated. Becausesomatic embryo derived plants appear phenotypically uniform and becausesomatic embryogenesis has been used in other crop species to regenerateplants from transformed sources, we propose the following:

1. To continue evaluation of somatic embryo derived plants.2. To develop technology to regenerate plants from additional cell

and organ sources.3. To concentrate specifically on sources which may play a role

in obtaining transformed plants.

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'l'able1. Procedures for obtaining somatic embryos and plants fromwalnut cotyledon tissue.

Procedure

Excise cotyledon

Conditioning mediumBasal medium

Select somatic embryosat cotyledon stage

Break apical dormancyLeaf and root growth

Transfer to peat plugs

Hardening of plants

Condition Time (weeks)

Aseptic techniqueDark

Dark

Dark, basal medium

6-12, after full bloom2-4

2-4, several transfers

2-4, repeated transfers

Cold, 2°C, dark

16-h photoperiod

1/2 strength basalNon-sterile, reduced

humidity soil

8-104-644

Total time . 32-40

Table 2. Somatic Embryo Regeneration

Table 3. Origin of somatic embryo derived plants 1

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Treatment Embryos Germinated SurvivingInitiated Embryos Plants in Soil

No. No. % total No. % total-Cold treatmentY 288 38 13 15 5No cold treatment 216 74 34 29 13Total 504 112 22 44 9

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YCold treatment = 8-10 wks at 4°C, dark

Origin number Origin number

Scharch Franquette (6) 55 J. hindsii 3P.I. 18256 (endosperm) 12 pterocarya sp x Gustine 11Early Ehrhart 3 Payne 4Other Franquettes 10 TOTAL 98

Iplants obtained from endosperm or cotyledons of , open pollinated seed.